The main goal of the proposed project is to further delineate the molecular mechanisms involved in mercury-induced immunotoxicity. Exposure to mercury at low levels may alter immune parameters before it causes any clinically apparent manifestations. Even subtle immune system alterations may indirectly affect ability to resist microbial infections or development of cancers. Previous studies have shown that Brown Norway (BN) rats are highly susceptible for the development of autoimmune disease conditions but the molecular mechanisms including the involvement of Th1 and Th2 responses are not fully delineated. In a preliminary study, we have shown predominance of Th2 type responses in Sprague Dawley (SD) rats given oral gavage of mercuric chloride. We propose to develop a comprehensive data base that is needed to clarify the Th1/Th2 cell responses in the development of mercury-induced immunotoxicity. In the proposed project, we will study the effects of methyl-mercury using an in vivo protocol with both BN and SD rats and an in vitro protocol using human peripheral blood lymphocytes. We will study the status of Th1 cytokines, interferon-gamma, Interleukin-2 (IL-2) and Th2 cytokines, IL-4 and IL-10. We will employ several different assays including: real-time quantitative RT-PCR, ribonuclease protection assay (RPM, ELISA, and ELISPOT. These different assays targeting the Th1/Th2 cell responses are expected to give us detailed information on the expression of the cytokines at the level of both transcription and protein production. It will also give us some information on the status of cells involved in the secretion of these cytokines. We will compare the immune cells of BN and SD rats as well as human cells for activation markers CD25 (Il-2R alpha) and MHC class II antigens, and for various apoptosis markers, FAS, FASL, bcl-x (L), bcl-x (5), bax, bcl-2, and caspases 1, 2, 3. We will compare serum samples of BN and SD rats for immune-mediated disease markers including anti-nuclear antibody (ANA), circulating immune complexes, and C-reactive protein. To study whether mercury-induced alterations of the immune responses adversely affect host disease resistance mechanisms, we will experimentally infect the rats with Pseudomonas aeruginosa and study the morbidity and mortality. These in vitro and in vivo data will help us further delineate the molecular mechanisms involved in mercury-induced immunotoxicity which may be useful for planning immunomodulatory and immunotherapeutic measures in subjects exposed to mercury.